Inclusion complexes of 3-morpholino-sydnonimine or its salts or its tautomer isomer, process for the preparation thereof, and pharmaceutical compositions containing the same

ABSTRACT

The invention relates to inclusion complexes of 3-morpholino-sydnonimine or its salts or its tautomer isomer, process for the preparation thereof and pharmaceutical compositions containing the same. 
     The inclusion complex of 3-morpholino-sydnonimine or its salt formed with cyclodextrin derivative is prepared by 
     a) reacting the 3-morpholino-sydnonimine or its salt in an aqueous medium with a cyclodextrin derivative and the complex is isolated from the solution by dehydratation, or 
     b) high energy milling of 3-morpholino-sydnonimine or its salt and a cyclodextrin derivative.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Phase of PCT/HU91/00013 filed Mar. 28,1991 and based upon Hungarian national application 1869/90 of Mar. 28,1990 (amended Jun. 27, 1990) under the International Convention.

1. Field of the Invention

The invention relates to the inclusion complexes of3-morpholino-sydnonimine (SIN-1) or its salts or its open ring "A" formtautomer (SIN-1A: N-nitroso-N-morpholino-amino-acetonitrile) formed withcyclodextrin derivatives, to the process of preparation and topharmaceutical compositions containing them as active ingredients. Theopen ring "A" form plays an important role, its formation is predominantfor NO (nitric oxide) radical liberation.

2. Background of the Invention

Molsidomin (N-ethoxycarbonyl-3-morpholino-sydnonimine, SIN-10) is awell-known antianginal agent. In therapy it is widely used for thetreatment and prevention of angina in the case of heart insufficiency,in the state of heart infarct. The therapeutic effect of Molsidomin canbe attributed mainly to its first active metabolite to the3-morpholino-sydnonimine, which originates mainly in the liver byenzymatic hydrolysis and by subsequent effect of the decarboxylaseenzyme. Thus the SIN-1 possesses a therapeutic advantage too compared toMolsidomin, that is its efficacy is quicker and more definite. Itsdisadvantage is that it can be used only in the form of intravenousinjections.

The sydnonimine derivatives are extremely light-sensitive. Upon toirradiation with artificial or natural light for only a short time theywill be decomposed quickly. In the course of photolysispharmacologically inactive, but harmless decomposition products as e.g.morpholine, ammonia, ethyl alcohol, carbondioxyde form. The aim of somepatents is the photostabilization of sydnonimine derivatives by theaddition of different additives. (European Patent Specification No.206219 and GFR Patent Specification No. 3.346.638).

The light-sensitivity can fairly be avoided in dry state and in solutionby suitable storage (in a well-closed dark flask wrapped with blackpaper).

It can be generally said that the sydnonimine derivatives are sensitiveto extreme pH values, and are decomposed quickly. The final product ofthe chemical and metabolitic decomposition of Molsidomin is the SIN 1C(cyanomethylene-amino-morpholine), an inactive metabolite. In the use ofSIN 1 as a medicament the problems of chemical stability are coming tothe fore. The substance decomposes quickly in a neutral-aqueoussolution, it is sensitive to the pH value and it is stable only in astrong acidic medium (pH 1-2). Above pH 2 it decomposes quickly, to theinactive metabolite SIN 1C, that is connected with the loss oftherapeutic activity. The SIN 1→SIN 1C transformation is the result ofbase catalyzed hydrolytic decomposition. The process is strictly pHdependent, e.g. to achieve a decomposition of 10% (SIN 1C) 53 sec. at apH value of 8, 15 hours at a pH of 6, 67 days at a pH of 4, and 13 yearsat a pH of 1-2 are necessary. (Chem. Pharm. Bull. 19/6 1079, 1971). SIN1 is also decomposed in a diluted aqueous solution stored in diffusedlighting in 1-3 days. The decomposition can be followed from the UVspectrum directly. The value of the absorption maximum is shiftedgradually from 291±1 nm to 278±1 while the value of absorption increasescontinuously, which is in harmony with the data of the literature: the λmax. value of SIN 1 is 291±1 nm, the specific absorption coefficientA^(1%) =520, while the SIN 1C λ max. is 278±1 nm, its molar absorptioncoefficient ε=17000 A^(1%) =1220.

    1 cm

SIN 1 applied orally is not effective; between pH 1-2 SIN 1 haspresumably a strongly ionized state of favourable chemical stability,which on the other hand is not favorable to the resorption from the G1tract. Moving along from the stomach at a more basic pH in the sense ofthe above mentioned facts SIN 1 is transformed quickly into the inactiveSIN 1C metabolite. The low dose of the SIN 1 applied is favorable tothis transformation as the hydrolytic decomposition in lowconcentrations (of μ/ml order) is still more definite.

SIN 1 is on the market in the form of its hydrochloride salt, forintravenous application in the form of a lyophilized powder ampoulecontaining 2 mg active ingredient and 40 mg of sorbite. The content ofthe ampoule has to be dissolved in 1 ml of distilled water before use.

OBJECT OF THE INVENTION

The object of the invention was to prepare such a new agent, by means ofwhich the SIN 1→SIN 1C transformation can be hindered even in aneutral-aqueous solution, and at a physiological pH value.

DESCRIPTION OF INVENTION

It is known that the complexation with cyclodextrin is suitable to thestabilization of different agents against heat, light and basic oracidic hydrolysis. (Szejtli: Cyclodextrin Technology, Kluwer, Dordrecht,1988., pages 211-217).

In the course of our investigations we have found, that thecyclodextrins have proved themselves effective to stabilize thedilute-aqueous solutions obtained from the SIN 1 injectable composition.

The interaction between SIN 1 and the cyclodextrins was proved by thefollowing test series:

From the injectable SIN 1 composition we prepared an aqueous solution ofabout 10 μg/ml and in the solutions 20-40 mg of cyclodextrin orcyclodextrin derivative were dissolved while stirring.

The solutions were stored at room temperature (20°-22° C.) in diffuselighting and in certain periods (daily) the UV spectrum of the solutionswere registered in the interval between 220-350 nm. As a control onlythe aqueous solution, and the 0,05N hydrochloric acid solution were usedrespectively. The pH value of the cyclodextrin solutions were checked todetermine, that cyclodextrin, and the derivative used, respectively, donot change significantly the pH value of water. The pH values of theapplied cyclodextrin solutions of greatest concentration differed onlywithin ±0.2 unit from the pH value of distilled water. We found that theshift of the UV spectrum was conspicously well hindered by CDPSI, anionic soluble β-cyclodextrin polymer (average molecule mass 3500, β-COcontent 54%, COO⁻ content: 4.2%) (Hungarian Patent Specification No.191.101). The shift of the spectrum was not experienced even after 2weeks of storage, while the aqueous solution stored similarly waspractically transformed into SIN 1C within 2-3 days.

The decomposition hindering, slow down effect of DIMEB(2,6-di-O-methyl-β-cyclodextrin) is definite too. In the presence of 20mg/ml DIMEB the spectrum of the solution has shown only a shift within 1nm after a 6 day storage. We also tested the stabilizing effect ofTRIMEB (2,3,6-tri-O-methyl-β-cyclodextrin), of γ-CO, β-CO and ofhydroxypropyl-β-CO. The efficiency sequence concluded from the UVspectrum shift was as follows:

    CDPSI>DIMEB>TRIMEB>βCO≧HPβCO.

From the effect exerted on the hydrolytic decomposition of SIN 1 by thedifferent cyclodextrins and outstandingly by CDPSI a complex interactioncan concluded.

In the above test series the cyclodextrins and their derivatives wereused in an extremely great excess (10 μg/ml of SIN 1 vs ˜20 mg/ml ofcyclodextrin).

The test series were repeated so, that CDPSI was applied used in a 10fold or 20 fold excess, which corresponds to a SIN 1: CDPSI weightproportion in a complex containing 10% or 5% respectively of the activeingredient. The low therapeutic dose of SIN 1 allows even theapplication of a complex containing less than 5% of the activeingredient, which corresponds to a molar ratio of about 1:2 SIN 1:CDPSI. In such a concentration range spectrophotometric measurement dosenot give a descriptive picture. For this reason the decomposition degreewas determined by thin-layer chromatography in the following way: Fromthe solutions 10 μl were dropwise added on a Kieselgel 60 F₂₅₄ plate(10×10 cm, Merck). Running mixture: cyclohexane:ethyl-acetate in a ratioof 1:1. The running tub was let for 30 minutes to saturation. On theplate also SIN 1C reference solution was dropwise added. On the droppingspot the plate was dried in a cold air flow protected from light and itwas let to run up to a 15 cm height. During the running period therunning tub was put on a dark place. After evaporation of the solventthe plate was evaluated visually in UV light at 254 nm. SIN 1 presentsat 0,05 R_(f) and SIN 1C at 0.36 R_(f). We experienced in every casethat in the solutions containing CDPSI the intensity of the SIN 1C spotis visibly smaller than in aqueous solutions.

As the transformation of SIN 1 to an inactive metabolite is considerablypH dependent, the measurements were carried out in phosphate buffers ofpH 6.4, 7.0 and 7.6 according to the pharmacopoea. In ever case thebuffer and 20 mg/ml CDPSI containing buffer solutions were compared, allof them by UV spectophotometric measurement after suitableaqueous-alcoholic dilution, examinating the intensity of the SIN 1decomposition product by thinlayer chromatography.

The results of the spectrophotometric measurement are summarized inTable I.

                  TABLE I                                                         ______________________________________                                        The change of the λ max. value as function of time (1 mg/ml            SIN 1 + 20 mg/ml CDPSI)                                                       λ max.                                                                 time  pH 6.4       pH 7.0       pH 7.6                                        (days)                                                                              buffer  +CDPSI   buffer                                                                              +CDPSI buffer                                                                              +CDPSI                              ______________________________________                                        at preparation                                                                0     291.5   291.9    290.6 291.0  291.2 290.9                               1     289.0   290.5    280.4 285.5  276.4 278.8                               4     284.9   288.5    --    --     --    --                                  6     279.8   283.9    277.3 278.8  277.6 277.9                               ______________________________________                                         The decrease of the maximum UV wavelength of about 13 nm (from 291 nm to      278 nm) represents the total decomposition of SIN 1 to SIN 1C.           

In the buffer of pH 6,4 in the presence of CDPSI still after 6 days theΔλ was =7 nm by contrast to the control Δλ=12 nm value. Thereproductibility of the UV maximum under the given circumstances waswithin ±0.5 nm, thus the difference can be considered as significant.After 1 day of storage the Δλ difference could be still measured, bycontrast at pH 7.6 the stabilization effect of CDPSI scarcely isdiscerned.

In the tested solutions the SIN 1: CDPSI molar ratio was about 1:2,which is the necessary minimum for the complexation in solution. Thestabilizing effect in solution of the 10 fold excess (10 mg/ml),-corresponding to a complex of 1:1 molar ratio--of CDPSI was scarcelypresent. The decomposition degree could be followed by thin-layerchromatography too. The SIN 1C spot intensity presented at 0.36 R_(f) ina pH 6.4 buffer differed still after 1 week of storage noticeably; in a7 and 7.6 buffer after 1 day of storage a perceptible difference wasshown in the intensity of the SIN 1 spot at 0.04 R_(f). In the pH 7buffer the unchanged SIN 1 was still perceptible, in that of 7.6 it waspractically no longer detectable.

In solution the equilibrium of the dissociation of the complex can beshifted, using the cyclodextrin in an excess. With an extremely greatCDPSI excess (1000, 2000 fold respectively) the injectable compositionhas shown only a spectrum shift of Δλ±1 nm in distilled water after 1week, while the control solution was practically decomposed.

The quantitive measurement of the decomposition degree was carried outby the HPLC method too.

HPPLC measurement circumstances to the separation of SIN 1-SIN 1 C

    ______________________________________                                        Equipment:                                                                            Beckman 114 M Solvent Delivery Module,                                        165 Variable Wavelength Detektor, Hewlett-Packard                             3396 A integrator.                                                    Column: Ultrasphere ODS analytical column 4.6 × 150 nm,                         5μ.                                                                Eluent: 0.05 M sodium acetate:                                                                         700 ml                                                       acetonitrile:    300 ml                                                       tetrahydrofuran:  2 ml                                                        flowing rate:     1.0 ml/min.                                                 pressure:        120 bar                                                      measuring wavelength:                                                                          278, 290 nm                                                  sample volume:    20 μl                                                    sensibility:      0.1 AU                                                      chart speed:      0.5 cm/min.                                                 retention times: t.sub.R SIN1: 2.9 min.                                                        t.sub.R SIN 1C: 4.2 min.                             ______________________________________                                    

Results:

1. Solutions of 0.5 mg/ml SIN 1 prepared in phosphate buffer of 6.3 pHprotected from light were stored at room temperature. Complex was formedwith 50 mg/ml (100 fold quantity) of CDPSI. After storage of 5 weeks theHPLC chromatograms were evaluated.

    ______________________________________                                        Evaluation:                                                                   Active ingredient                                                             content:       control.sup.x                                                                          CDPSI-complex                                         ______________________________________                                        SIN 1          0,016    0,17                                                  SIN 1C         0,23     0,16                                                  ______________________________________                                         .sup.x Note: The data of the control measurement relates to distilled         water and not to buffer.                                                      CDPSI applied in a 100 fold weight excess (about in a molar ratio of 10:1     is hinders significantly the SIN 1 decomposition. The result obtained is      in accordance with the thinlayer-chromatography test.                    

2. The test was carried out in a pH 7 phosphate buffer too, choosingsimilar concentration circumstances. (0,5 mg/ml SIN 1 50 mg/ml CDPSI).

The chromatograms obtained after 4 days storage were evaluated.

    ______________________________________                                        Evaluation:                                                                   Active ingredient                                                             content mg/ml  control  CDPSI-complex                                         ______________________________________                                        SIN 1          0.041    0.23                                                  SIN 1C         0.125    0.064                                                 ______________________________________                                         pH value of the solutions after storage:                                      Control: 6.57                                                                 CDPSI: 6.2                                                               

It can be considered as proved, that in the concentration used CDPSIhinders even at pH 7 the transformation of SIN 1 to SIN 1C which is aninactive metabolite.

We tested further the stability of SIN 1 injection in a dilute aqueoussolution.

From the lyophilized SIN 1 powder ampoule an aqueous solution of 50μg/ml concentration was prepared, thereafter 20 mg/ml of CDPSI wereadded. The solutions were stored at room temperature, and from time totime the SIN 1C decomposition product quantity was determined.

The chromatograms obtained after the 1.; 4. and 11. days were evaluated.

    ______________________________________                                        Evaluation:                                                                               SIN 1C content μg/ml                                           time (days)   control CDPSI-complex                                           ______________________________________                                        starting      0.1     0.1                                                      1 day        0.8     0.6                                                      4 days       3.9     0.94                                                    11 days       5.6     1.8                                                     ______________________________________                                    

Thus object of the present invention is a SIN 1 cyclodextrin derivativeand the preparation thereof, which is stable in an aqueous system evenat physiological pH and in which the transformation to a SIN 1C inactivemetabolite is hindered by the applied cyclodextrin derivative.

Surprisingly it was found that with cyclodextrins, especially with CDPSIcomplexed SIN 1 contains a considerable amount of SIN 1A intermediateafter the complex preparation. The transformation process of SIN 1→SIN1A→SIN 1C in the presence of cyclodextrins was also examined in 0.02Macetate buffer at pH 5.5. Surprisingly the effect of β-CO was the mostpronounced, more than 7 fold more SIN 1A was found in the presence ofβ-CO than in the control. (The SIN 1A was detected by HPLC)

Testing the biological effect of the compositions according to theinvention in vivo, we found that till SIN-1 used p.o. in a dose of 1mg/kg does not exert an activity (cardioprotective activity on rats is11%), the cardioprotective activity of SIN-1-CDPSI applied p.o. with thesame active ingredient is 42,2%.

The good biological effectiveness of SIN 1--CDPSI complex in thecardioprotective test after oral administration can be attributed to SIN1A present in the solid complex. The "A" form which plays a key role inthe biological effect, cannot be formed under the pH of gastric medium;it can be generated in-vitro from SIN 1 substance by incubation underbelow alkaline pH. Upon dissolving the CDPSI complex in distilled waterconsiderable amount of SIN 1A can be detected in the aqueous solution.

It seems that the transition of SIN 1-SIN 1A is promoted bycyclodextrins, and simultaneously the very unstable, oxygen sensitiveSIN 1A is stabilized by complexation, which results in slower SIN 1A→SIN1C transformation. Thus the smoother onset and the longer duration ofaction are due to a delayed release of nitric oxide from complexed SIN1.

The following TABLE shows the SIN 1A content of SIN 1 complexes withdifferent cyclodextrins prepared according to Example 2 measured rightafter the preparation by HPLC. SIN 1 physical mixture at the samecomposition prepared in the same manner with lactose was used asreference.

                  TABLE                                                           ______________________________________                                        (Sample)         SIN 1A content %                                             Complex          (given in SIN 1C equivalent)                                 ______________________________________                                        SIN 1 - CDPSI    1.27                                                         SIN 1 - β-CO                                                                              0.08                                                         SIN 1 - DIMEB    0.06                                                         SIN 1 - HPβCO                                                                             0.115                                                        SIN 1 - lactose reference                                                                      --                                                           ______________________________________                                    

Results and conclusions

The relaxing effects of the SIN 1-cyclodextrin complexes and of normalSIN 1 were studied in the concentration 1 or 2 μM.

All four complexes relaxed the sustained contraction of potassiumdepolarized strips with a maximal effect of 43% to 53%. SIN 1 (1 μM) wassomewhat more effective with a relaxation of 58%.

Maximal relaxation occurred with the SIN 1 complexes 23 min to 32 minafter administration whereas T/max of SIN 1 was at 18 min. Thisdifference was statistically significant. The duration of action asmeasured by T/2 was longer with the cyclodextrin-complexes (62 min to 88min) than with SIN 1 (47 min). This difference was also statisticallysignificant. Thus, the SIN 1-cyclodextrin-complexes showed a smootheronset and a longer duration of action than normal SIN 1.

Since the relaxing effect of SIN 1 is thought to be due to nitric oxidereleased by an oxidative decomposition process it may be concluded thatthe complexation of SIN 1 with cyclodextrins slows down thisdecomposition process. Thus, the smoother onset and the longer durationof action are likely due to a delayed release of nitric oxide from SIN1-cyclodextrin-complexes.

According to the facts set forth above the invention within inclusioncomplexes of 3-morpholino-syndonimine or of its salts or its open ring"A" form tautomer isomers are formed with cyclodextrin derivatives.

As cyclodextrin derivative the inclusion complexes according to theinvention contain advantageously an ionic, water-soluble cyclodextrinpolymer (CDPSI) (molecular weight <10000),heptakis-2,6-dimethyl-β-cyclodextrin (Dimeb),heptakis-2,3,6-tri-O-methyl-β-cyclodextrin (Trimeb) and βorγ-cyclodextrin.

To the preparation of a solid inclusion complexhydroxypropyl-β-cyclodextrin can be used too.

The inclusion complexes according to the invention are prepared byreacting the 3-morpholino-sydnonimine or its salts, in a solvent mediumwith a cyclodextrin derivative and if desired the complex is obtainedfrom the solution by dehydratation, or by high energy milling of the3-morpholino-sydnonimine or its salts and the cyclodextrin derivative.

The complex can be advantageously isolated from the solution bylyophilization, spray-drying, evaporation in vacuum at low temperatureand by vacuum drying.

The SIN 1 is dissolved with 1-40 mmoles of CDPSI, or Dimeb in 1-500 mldistilled water calculated for 1 mmole of active ingredient, thereafterthe dehydration is carried out as mentioned before. The molar ratio ofCDPSI polymer is calculated on β-CO. Thus applying the polymer of about50% β-CO content and of the average 3500 molecular mass the compositionof the complex of 1:1 ratio corresponds to about 8%, and and that of 2:1molar ratio to about 4.5%.

Complex interaction in solution was illustrated by the membranepermeation test

Visking type cellophane membrane (average pore diameter 24 Angstroms)was used. Aqueous solution of SIN 1 at a concentration of 1 mg/ml wereput in the donor cell, while distilled water was placed in the receptorcompartment of the membrane permeation cell apparatus. The solutionswere stirred by magnetic stirrers and were kept at 37°±1° C. Atappropriate time intervals samples were taken from the receptor solutionand concentration of SIN 1 had permeated from the donor cell wasmeasured by UV-spectrophotometry. The test was repeated in the presenceof different cyclodextrins at different concentrations in the donor cellcompartment. The time required for the diffusion of 50% SIN 1 (T₅₀ %) inthe presence of CDPSI at different concentration are listed in theTABLE.

    ______________________________________                                        Diffusion half life of SIN 1 in the presence                                  of CDPSI                 .sup.T 50% .sup.(hours)                              ______________________________________                                        SIN 1 alone              0.9                                                  +CDPSI 25 mg/ml          3.5                                                  50 mg/ml                 5.0                                                  ______________________________________                                    

The inclusion complexes prepared according to the invention can be usedto the production of pharmaceutical compositions, combinationsrespectively for stable injectable, oral or local use.

Doses of the inclusion complexes of this invention may vary with theage, body weight and conditions of the subject, the administrationroute, the number of administrations or the like, but is in the range of6 to 800 mg per day, perferably 10 to 400 mg per day.

The retard effect prevails particularly in the case of applying thepharmaceutical composition in the form of one-a-day tablets,microcapsules and ointments exceedingly suitable for percutaneous use,respectively. The pharmaceutical compositions of the invention areprepared in a customary manner. The adjuvants and carriers are thosewhich are usually used in the field of pharmaceutical preparations.

The details of the invention are illustrated below in some exampleswithout limiting the invention to the examples.

EXAMPLES 1. Preparation of the SIN-1-CDPSI complex by lyophilization

15 g of (6.6 mmoles) CDPSI polymer are dissolved in 200 ml of distilledwater, thereafter to the solution 1.1 g (5.3 mmoles) of SIN 1-HCl areadded. The materials are dissolved almost immediately and a pure, clearsolution is obtained, which is instantly frozen and dehydrated bylyophilization, taking care, that in the course of any processes thesubstance should be exposed to the possible minimal light exposure.

It is e.g. expedient to wrap up the alembic with a black paper whiledissolving. The product obtained is very light, it is a loose powder,its active ingredient content determined by a spectrophotometric methodis: 6.5±0.5%, corresponding about to a molar ratio of 1:1.

Tests proving the fact of complex formation: the thermogravimetric (TG),differential-scanning colorimetric (DSC) and the Thermal EvolutionAnalysis (TEA) tests have shown characteristic differences between SIN 1the physical mixtures of SIN 1-CDPSI and SIN 1-CDPSI complex. From theSIN 1 active agent at a temperature of 60°-110° C. about 8% of aninorganic substance is removed; it is possible that it is water. Thedecomposition while melting of the substance started at a temperature of170°-180° C. with an explosive violence and in a very tight temperatureinterval 70% of the introduced agent is removed from the system.

Between 220° and 230° C. the decomposition slows down, and, up to 350°C. a 87% loss of the mass can be register. To the identification of thenon-complexed active ingredient the DSC peak between 190°-200° C. andthe 190° C. TEA peak causing even in an argon atmosphere an exothermenthalpy alteration can be used. The thermic curves of the SIN 1-CDPSIphysical mixture prepared immediately before measuring can be consideredas resultants of the starting materials.

The curves of the complex show a significant discrepancy from thoseabove, the pointed decomposition peaks of SIN 1 do not representthemselves, which means, that SIN 1 really forms an inclusion complexwith CDPSI.

Powder X-ray diffraction pattern

According to SIN 1-CDPSI X-ray diffraction tests, the structure is anamorph one. The characteristic reflection peaks of SIN 1 aredisappearing. The amorphons structure of CDPSI is known. Thecrystallisation degree of SIN 1 treated similarly but without CDPSI isthough decreasing, but the transition to a total amorphons structure isnot possible only by lyophilization. 2 ○ ˜ reflection peaks hinting aslightly crystalline form, characteristic to free (not complexed) SIN 1can't be found in the complex diagram.

2. Preparation of the SIN 1-CDPSI complex by lyophilization

15 g of CDPSI polymer (6.6 mmoles) (β-CO content 53%, COO⁻ content 4.2%)are dissolved in 200 ml of distilled water. In the solution obtained 0.7g (3.3 mmoles) of SIN 1 are dissolved, thereafter the solution isprocessed according to example 1.

Active ingredient content of the product obtained is 4.5±0.2%,corresponding about to a 1:2 molar ratio.

3. Preparation of SIN 1-CDPSI complex with high and controlled SIN 1Acontent

Following the process according to Example 2 the obtained solid productis submitted to a second drying to remove the complex bound water. Theproduct was dried at 60° C. under vacuo for 3 hours. (to a constantweight) Loss on drying: 4.5±0.2%. SIN 1A content was increased aboutfive fold during heating, it increased from 1.08% to 5.1% while the SIN1A/SIN 1C ratio was also altered favorably. (changed from 10 to 17.)Storing the dried complexes at room temperature protected from lightmore than 3 months the SIN 1A content and the ratio of SIN 1A/SIN 1C ofthe sample practically was not changed. In the control (notheat-treated) sample during storage the SIN 1A content is also increasedfrom 1.08% to 1.9%, at the same time the ratio of SIN 1A/SIN 1Cdecreased (changed from 10 to 3). Preparation of SIN 1-CDPSI complexproduct with high SIN 1A content and controlled composition is possibleby a short term heat-treating of the lyophilized SIN 1-CDPSI complex.

4. Preparation of SIN 1 Dimeb complex:

14 g of Dimeb (10.3 mmoles) (moisture content 2%) are dissolved in 100ml of distilled water. While stirring the solution 1.03 g (5 mmoles) ofSIN 1 are added. The clear solution obtained is processed according toexample 1 protected from light. The product is a loose white powder,active ingredient content is 6.5±0.2%, the molar ratio corresponds aboutto 1:2 SIN 1: Dimeb.

5. Preparation of an ointment for percutaneous use with a SIN 1 contentof 10 mg and ointment content of 1/2 g

From the SIN 1-Dimeb complex prepared according to example 3 (activeingredient content 6.5%) 151 mg are dissolved in 20 ml of distilledwater. To the solution protected from light 50 mg of KLUCHEL-HF(hydroxypropylcellulose) are added while vigorous stirring. Thus aviscous solution, hardly miscible is obtained, it is let to stay at roomtemperature for one day. Thus a transparent jelly is formed, 2 g ofwhich are containing 10 mg of SIN 1.

6. SIN 1 tablet with 2 mg active ingredient content/tablet

80 mg of SIN 1-CDPSI complex, active ingredient content 2.5%,

40 mg of maize startch,

128 mg of milk sugar

2 mg of magnesiumstearate

total weight of the tablet: 250 mg

The tablets are prepared by the method known per se, by direct pressure.

What we claim is:
 1. An active inclusion complex of3-morpholino-sydnonimine or of its pharmaceutically acceptable salt ortautomer isomer formed with a cyclodextrin.
 2. Inclusion complex as inclaim 1 of 3-morpholino-sydnonimine or of its pharmaceuticallyacceptable salt or tautomer isomer formed with an ionic water solublecyclodextrin polymer CDPSI, having a molecular weight <10,000. 3.Inclusion complex as in claim 1 of 3-morpholino-sydnonimine or of itspharmaceutically acceptable salt or tautomer isomer formed withhydroxypropyl-β-cyclodextrin.
 4. Inclusion complex as in claim 1 of3-morpholino-sydnonimine or of its pharmaceutically acceptable salt ortautomer isomer formed with heptakis-2,6-dimethyl-β-cyclodextrin. 5.Inclusion complex as in claim 1 of 3-morpholino-sydnonimine or of itspharmaceutically acceptable salt or tautomer isomer formed withheptakis-2,3,6-tri-0-methyl-β-cyclodextrin.
 6. Inclusion complex as inclaim 1 of 3-morpholino-sydnonimine or of its pharmaceuticallyacceptable salt or tautomer isomer formed with β- or γ-cyclodextrin. 7.Process for the preparation of the active inclusion complex ofN-nitroso-N-morpholino-amino-acetonitrile or its pharmaceuticallyacceptable salt formed with a cyclodextrin-derivative, which comprisesa)reacting the 3-morpholino-sydnonimine or its pharmaceutically acceptablesalt in an aqueous medium with a cyclodextrin derivative and the complexis isolated from the solution by dehydration, or b) high energy millingof the 3-morpholino-sydnonimine or its salt and a cyclodextrinderivative, whereby the complexed 3-morpholino-sydnonimine undergoes atransformation to form the N-nitroso-N-morpholino-amino-acetonitrile. 8.Process according to claim 7, wherein as cyclodextrin derivative anionic, water soluble cyclodextrin polymer (molecular weight<10.000);hydroxypropyl-cyclodextrin, heptakis-2,6-O-dimethyl-β-cyclodextrin,heptakis-2,3,6-tri-O-methyl-β-cyclodextrin or β-cyclodextrin are used.9. Pharmaceutical composition, containing as active ingredient atherapeutically effective amount of the inclusion complex of claim 1 or3-morpholino-sydnonimine or of its pharmaceutically acceptable salt, ortautomer isomer formed with a cyclodextrin together with apharmaceutically acceptable inert carrier.
 10. Pharmaceuticalcomposition according to claim 9, formulated in an oral form. 11.Pharmaceutical composition according to claim 9, which contains as acyclodextrin an ionic, water soluble polymer CDPSI having a molecularweight<10,000; hydroxypropyl-β-cyclodextrin,heptakis-2,6-O-dimethyl-β-cyclodextrin,heptakis-2,3,6-tri-O-methyl-β-cyclodextrin, β-cyclodextrin orγ-cyclodextrin.
 12. Method of treatment of anginic or ischemic diseasein a human in need of said treatment which comprises administering tothe human a therapeutically effective amount of an active inclusioncomplex of 3-morpholino-sydnonimine or its pharmaceutically acceptablesalt or tautomer thereof formed with a cyclodextrin as defined inclaim
 1. 13. An active inclusion complex ofN-nitroso-N-morpholino-amino-acetonitrile or of its pharmaceuticallyacceptable salt formed with a cyclodextrin.
 14. The inclusion complexdefined in claim 13 of N-nitroso-N-morpholino-amino-acetonitrile or ofits pharmaceutically acceptable salt with an ionic water-solublecyclodextrin polymer CDPSI having a molecular weight<10,000.
 15. Aprocess for the preparation of an active inclusion complex ofN-nitroso-N-morpholino-amino-acetonitrile or of its pharmaceuticallyacceptable salt formed with a cyclodextrin, which comprises the stepsof:(a) complexing 3-morpholino-sydnonimine or a pharmaceuticallyacceptable salt thereof with the cyclodextrin in an aqueous medium andisolating by dehydration the cyclodextrin inclusion complex of the3-morpholino-sydnonimine or the pharmaceutically acceptable salt thereofthus formed as a solid product; and (b) subjecting the cyclodextrininclusion complex of the 3-morpholino-sydnonimine or thepharmaceutically acceptable salt thereof to a subsequent drying step at60° C., under vacuum for 3 hours, to remove bound water from saidinclusion complex and to form the cyclodextrin inclusion complex ofN-nitroso-N-morpholino-amino-acetonitrile or the pharmaceuticallyacceptable salt thereof.
 16. The process defined in claim 15 wherein thecyclodextrin is an ionic, water-soluble cyclodextrin polymer CDPSIhaving a molecular weight<10,000.
 17. The process defined in claim 15wherein according to step (a) the dehydration of the cyclodextrininclusion complex of the 3-morpholino-sydnonimine or thepharmaceutically acceptable salt thereof is carried out bylyophilization.